基于Wnt/β-catenin信号通路探讨雷公藤甲素联合脉冲射频对大鼠神经性疼痛模型抗炎镇痛的作用机制

Protective role of triptolide combined with pulsed radiofrequency in anti-inflammatory and analgesic effects via Wnt/β-catenin signaling pathway

目的 探讨雷公藤甲素联合脉冲射频对大鼠神经性疼痛模型的抗炎镇痛作用及相关机制。方法 将SD大鼠随机分为假手术组、脊神经结扎组、雷公藤甲素组、脉冲射频组、雷公藤甲素联合脉冲射频组。构建脊神经结扎模型大鼠,检测大鼠疼痛行为学变化;蛋白质印迹法检测坐骨神经组织中Wnt-3α、β-catenin、frizzled7蛋白表达;聚合酶链式反应检测坐骨神经组织中Il-1β、Cox-2、Tnf-α、p65 Nf-κB mRNA表达;免疫组织化学检测坐骨神经组织中SP和C-fos蛋白表达。结果 除假手术组外,其余各组机械性缩足反射阈值在术后第1天急剧下降,术后第5天脊神经结扎组的机械性缩足反射阈值趋于稳定,各治疗组的机械性缩足反射阈值逐渐上升。除假手术组外,其余各组热缩足反射潜伏期在术后前3天急剧下降,术后第3天各组热缩足反射潜伏期开始升高,各治疗组的升高速度均高于脊神经结扎组。蛋白质印迹法结果显示术后第3、7、14天,脊神经结扎组Wnt-3α、β-catenin、frizzled7蛋白表达升高。与脊神经结扎组比,术后第3天各治疗组Wnt-3α、β-catenin、frizzled7蛋白表达差异较小,术后第7天和第14天各治疗组蛋白表达均降低,且雷公藤甲素联合脉冲射频组的相对表达量最低。聚合酶链式反应结果显示术后第3、7、14天,与假手术组比,脊神经结扎组的Il-1β、Cox-2、Tnf-α、p65 Nf-κB mRNA表达升高。与脊神经结扎组比,术后第7天和第14天各治疗组的mRNA表达均降低,且雷公藤甲素联合脉冲射频组表达最低。免疫组织化学结果显示脊神经结扎组中SP和C-fos蛋白表达高于假手术组,且随着时间的延长两种蛋白表达逐渐升高。与脊神经结扎组比,各治疗组SP、C-fos蛋白表达水平随治疗时间的延长均降低,且雷公藤甲素联合脉冲射频组降低最明显。结论 雷公藤甲素联合脉冲射频治疗通过调控Wnt/β-catenin信号通路减轻大鼠神经性疼痛模型炎症和疼痛反应。

Objective To investigate the anti-inflammatory and analgesic effects of triptolide combined with pulsed radiofrequency(RF)on neuropathic pain in rat models and related molecular mechanisms.Methods SD rats were randomly divided into a sham surgery group,spinal cord nerve ligation group,trip-tolide group,pulse RF group and triptolide combined pulse RF group.Spinal cord nerve ligation-model rats were cultivated and pain behavior changes in them measured.The expressions of Wnt-3α,β-catenin and friz-zled7 in sciatic nerve tissue were measured by Western blotting,mRNA expression of Il-1β,Cox-2,Tnf-αand p65 Nf-κB in sciatic nerve tissue measured by polymerase chain reaction and expression of SP and C-fos protein in sciatic nerve tissue detected by immunohistochemistry.Results Except for the sham surgery group,the mechanical foot contraction reflex threshold of the other groups sharply decreased on the first day after surgery;on the fifth day,the mechanical foot contraction reflex threshold of the spinal cord nerve ligation group tended to stabilize,while that of each treatment group gradually increased.Except the sham surgery group,the thermal shrinkage reflex latency of the other groups decreased sharply on the first 3 days after sur-gery,and began to increase on the third day,and the rate of increase in all treatment groups was higher than that in the spinal cord nerve ligation group.The results of the protein immunoblotting showed that,on postop-erative day 3,7 and 14,the expression of Wnt-3α,β-catenin,and frizzled7 proteins in the spinal cord nerve ligation group was higher compared to the sham surgery group.Compared to the spinal cord nerve ligation group,the differences in the expression of Wnt-3α,β-catenin,and frizzled7 proteins between the treatment groups were relatively small on postoperative day 3,but decreased in all treatment groups on pday 7 and 14,with the combined treatment of triptolide and pulse radiofrequency group showing the lowest relative expres-sion level.The results of polymerase chain reaction analysis revealed an increase in the expression of Il-1β,Cox-2,Tnf-α,and p65 Nf-κB mRNA in the spinal cord nerve ligation group compared to the sham surgery group on postoperative days 3,7 and 14.Compared to the spinal cord nerve ligation group,the mRNA expres-sion in all treatment groups decreased on postoperative days 7 and 14,with the combination of triptolide and pulsed radiofrequency group showing the lowest expression level.The immunohistochemical results showed that the protein expression of SP and C-fos in the spinal cord nerve ligation group was higher than that in the sham surgery group,and the expression of the two proteins increased gradually with the extension of time.Compared with the spinal cord nerve ligation group,the protein expression levels of SP and C-fos in all treatment groups decreased with the prolongation of treatment time,and the decrease was most obvious in triptolide combined pulse RF group.Conclusion Triptolide combined with pulsed radiofrequency treatment alleviates inflammation and pain response in a rat model of neuropathic pain by regulating the Wnt/β-catenin signaling pathway.